JP2009120562A - Mycosporin-like amino acid derivative having glycosyl group and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mycosporin-like amino acid derivative having ultraviolet absorbing action and a moderate moisturizing action, a method for producing the mycosporin-like amino acid derivative in high efficiency, an ultraviolet absorber containing the mycosporin-like amino acid derivative, especially an ultraviolet absorber for cosmetic use, and an ultraviolet protecting cosmetic containing the ultraviolet absorber. <P>SOLUTION: The mycosporin-like amino acid derivative has glycosyl group, and the ultraviolet absorber contains the mycosporin-like amino acid derivative. In the method for producing the ultraviolet absorbing substance containing a mycosporin-like amino acid derivative having glycosyl group by a step to culture an algae belonging to the genus Nostoc, order Nostocales, class Cyanobacteria under irradiation of active energy ray, the actinic energy ray irradiation is performed by the irradiation with ultraviolet rays of 280-315 nm wavelength at an irradiation intensity of 1.5-4.0 W/m<SP>2</SP>. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a mycosporin-like amino acid derivative and a method for producing the same, and various additives using the mycosporin-like amino acid derivative, particularly an ultraviolet absorber. The mycosporin-like amino acid derivative having a glycosyl group of the present invention has an absorption maximum at a wavelength of 320 nm to 330 nm and has a moisturizing action, and can be preferably used for cosmetics and the like.

  Sun rays are mainly classified into ultraviolet rays, visible rays, and infrared rays. Many of these sunlights have been reported to have various effects on the human body. As for ultraviolet rays, most of them are bad effects, and examples thereof include photoaging such as spots and wrinkles, DNA damage, skin cancer and the like. In this way, with the close-up of the adverse effects of ultraviolet rays on the human body, ultraviolet protective cosmetics using ultraviolet absorbers have attracted attention as a method for efficiently protecting the human body from ultraviolet rays.

  Conventional UV absorbers used in UV protective cosmetics include synthetic products such as benzophenones and octyl motoxycinnamate, all of which are concerned about safety such as cytotoxicity. Therefore, mycosporine-like amino acid derivatives have attracted attention as substances having a naturally occurring ultraviolet absorbing effect (see Patent Documents 1, 2, and 3).

  Mycosporine-like amino acid derivatives (mycosporine-like amino acids) have a partial structure of mycosporin (structure in which amino acid or amino alcohol is bound to cyclohexenone or cyclohexenimine skeleton) produced by seaweed, marine animals, microalgae, fungi, etc. A general term for amino acids. Mycosporine-like amino acid derivatives are known to have a strong absorption band from the UVB region (wavelength 280 to 315 nm) to the UVA (wavelength 320 to 400 nm) region.

JP-A-6-062878 JP-A-10-77472 JP 2007-16004 A

  However, the present inventors have found that the moisturizing action is low even when the mycosporin-like amino acid derivative is used for cosmetics. In general, when the moisturizing action is low, it causes dryness of the skin, and not only stains, wrinkles, and rashes are likely to occur, but also the skin secretes sebum to compensate for dryness, so that makeup collapse tends to occur. For this reason, when the mycosporin-like amino acid derivative is used as a UV absorber in cosmetics, a moisturizing conditioner must be added separately with the UV absorber, which may lead to an extra cost. there were.

  Accordingly, an object of the present invention is to provide a mycosporin-like amino acid derivative having an ultraviolet absorbing action and an appropriate moisturizing action, a method for efficiently producing the mycosporin-like amino acid derivative, and the mycosporin-like amino acid. An object of the present invention is to provide an ultraviolet absorber containing a derivative, particularly an ultraviolet absorber for cosmetic use, and an ultraviolet protective cosmetic containing the ultraviolet absorber.

As a result of diligent research to solve the above-mentioned problems, the present inventors have cultivated the cyanobacteria Nemoptera Nostoc, extracted from the culture and purified, and the mycosporin-like amino acid derivative has excellent ultraviolet absorption. It has been found that it has not only an action but also an appropriate moisturizing action, and the present invention has been completed. That is, the present invention
(1) The following general formula (I)

(Wherein R represents a glycosyl group) a mycosporin-like amino acid derivative having a glycosyl group represented by:
(2) an ultraviolet absorber containing the mycosporin-like amino acid derivative,
(3) A method for producing an ultraviolet-absorbing substance comprising a mycosporin-like amino acid derivative having a glycosyl group, which comprises a step of culturing an algae belonging to the genus Cyanobacteria Nostocidae while irradiating active energy rays, the active energy rays Provides a method for producing the ultraviolet absorbing material, characterized by irradiating ultraviolet rays having a wavelength of 280 to 315 nm with an irradiance of 1.5 to 4.0 [W / m ² ].

  According to the present invention, a mycosporin-like amino acid derivative having an ultraviolet absorbing action and an appropriate moisturizing action can be obtained. Furthermore, the mycosporin-like amino acid derivative can be produced efficiently. Further, the mycosporin-like amino acid derivative can be used as an ultraviolet absorber for various uses including cosmetics.

  The contents of the present invention will be described in detail below.

I. Method for producing ultraviolet absorbing material Culture Algae belonging to the genus Nostoc used in the present invention (hereinafter sometimes simply referred to as “Nostoc”) is a microorganism belonging to the genus Nostoc of the order Cyanobacteria and Nostocales, for example, Nostoc linkia, No. commune, N. muscorum, N. verrucosum and the like. Among them, Nostok linkia is preferable because it can produce a large amount of the mycosporin-like amino acid derivative having a glycosyl group of the present invention. Is a storage strain of a storage facility).

In general, Nostok not only has the ability to fix carbon dioxide by photosynthesis and the ability to fix air nitrogen, but also has resistance to drought, ultraviolet light, and oxidative stress. Therefore, it can grow not only in water but also on land, and is known as terrestrial cyanobacteria.
Therefore, the culture described above can be performed in water or in a mat-like state on agar containing water or soil, but water is preferred for industrial production.

  To cultivate Nostok in water, it can be carried out by a conventional method for culturing microalgae under a certain stirring condition under light irradiation in a medium of inorganic nutrient salts.

That is, the medium used for the culture is not particularly limited as long as it is used when culturing freshwater cyanobacteria, and examples thereof include MDM medium (Modified Detter medium). It is done. MDM medium consists of KNO ₃ 100 mg, MgSO ₄ .7H ₂ O 25 mg, K ₂ HPO ₄ 25 mg, NaCl 10 mg, CaCl ₂ .2H ₂ O 1 mg, Fe solution 0.1 ml, A5 solution 0.1 ml, water 99.8 ml. Become. The Fe solution consists of 200 mg of FeSO ₄ · 7H ₂ O, 100 ml of distilled water, 0.026 ml of concentrated sulfuric acid, and the A5 solution consists of 286 mg of H ₃ BO ₃ , 250 mg of MnSO ₄ · 7H ₂ O, ZnSO ₄ · 5H ₂ O 22.2. 2 mg, CuSO ₄ · 5H ₂ O 7.9 mg, Na ₂ MoO ₄ · 2H ₂ O 2.1 mg, distilled water 100 ml.

Since Nostok has the ability to fix nitrogen in the air, it is not always necessary to add a nitrogen source to the medium. However, in order to improve the growth rate, a method of adding a nitrogen source such as KNO ₃ like an MDM medium. Is preferred.

Nostok in water is added to the above medium so that the algae concentration (hereinafter sometimes simply referred to as “algae concentration”) is 0.05 to 0.1 g / L. It is carried out using a culture vessel.
The pH of the culture solution may be any of the known and conventional ranges as long as it is a pH at which Notok can grow, and of these, pH 5.0 to pH 10.0 is preferable, and pH 6.0 to 9.0 is particularly preferable. The pH of the culture solution may be adjusted by adding a buffer solution to the medium. However, as the amount of carbon dioxide in water decreases due to photosynthesis as the growth of Nostok proceeds, the pH increases, so aeration of carbon dioxide is performed. It is preferable to adjust by this. The carbon dioxide aeration method may introduce air mixed with carbon dioxide gas by an appropriate aeration means, or when it reaches a predetermined pH or more in conjunction with a pH meter, the carbon dioxide gas is automatically aerated directly. May be controlled.

  The temperature of the culture solution may be any known and conventional range as long as it is a temperature at which Nostock can grow, but is preferably 10 to 40 ° C, and particularly preferably 20 to 30 ° C.

  In addition, Notok is cultured while being irradiated with light. The light irradiation may be either artificial light or sunlight, but it is preferable to use artificial light to adjust the irradiance when irradiating active energy rays. When irradiating the active energy ray of artificial light, the irradiation is performed using a predetermined device, but otherwise, the artificial algae or sunlight that is capable of normal photosynthesis by the microalgae is irradiated. In order to increase the content of the UV-absorbing substance, (i) in the first culturing period, the concentration of algae in the medium is increased and light irradiation to the extent used in normal microalgae culturing is performed. It is preferable to irradiate active energy rays to increase the ultraviolet intensity of the irradiated light to a predetermined intensity.

  When light irradiation is performed with artificial light, the light source may be any light source capable of irradiating light with a predetermined wavelength and a predetermined intensity, such as a fluorescent lamp or a positive lamp. You may culture | cultivate using the fluorescent lamp normally used for culture | cultivation of a micro algae, for example, Sanyo FL40SS-VV / 37, or irradiating sunlight in the first culture period. In the later stage of the culture, irradiation with active energy rays is performed using a lamp capable of irradiating ultraviolet rays in the UVA region, for example, Toshiba FL40S-BLB-A, a lamp capable of irradiating ultraviolet rays in the UVB region, such as Sankyo Electric GL40SE. To do.

When irradiating an active energy ray, the ultraviolet intensity (irradiance) to be irradiated can be measured using a device that measures the ultraviolet intensity in the UVA and UVB regions, for example, a digital ultraviolet intensity meter (manufactured by Custom Inc.). The irradiance may be any irradiance that improves the content of the ultraviolet absorbing substance without killing the algae, but the irradiance of ultraviolet (UVB) is 0.5 to 5.0 (W / m ² ). It is preferable that it is 1.5-4.0 (W / m < ² >). Although the irradiation time varies depending on the intensity of ultraviolet rays to be irradiated, when the UV exposure amount of ultraviolet rays (UVB) is 30,000 to 400,000 (J / m ² ), it is preferably 1 to 72 hours, particularly 6 to 48 hours. preferable.

  The method for extracting a UV-absorbing substance and further a mycosporin-like amino acid derivative having a glycosyl group of the present invention from algae obtained after the culture (hereinafter sometimes referred to as “cultured alga bodies”) is generally microbial metabolism. The product can be performed according to a method commonly used for extracting and purifying the culture from the culture.

  For example, the culture is solid-liquid separated by filtration or centrifugation to harvest cultured alga bodies, and the algal bodies are extracted with an alcohol such as methanol or ethanol to obtain an extract containing an ultraviolet absorbing substance. Subsequently, the mycosporin-like amino acid derivative can be obtained by concentrating the extract and subjecting it to a column chromatography treatment such as a synthetic adsorption resin.

  Cultured algal bodies can be harvested by separating the culture solution and algal bodies by a filtration method using a filter cloth or by centrifugation.

  The harvested cultured algal bodies are subjected to an extraction process. At that time, as the form of algal bodies used for extraction, raw algal bodies, dried algal bodies, processed algal bodies processed by a method such as mechanical treatment, etc. Can be mentioned. Among these, the raw alga is preferable because it holds the ultraviolet absorbing substance more.

  Raw alga bodies are usually suspended in water depending on the degree of water removal when harvesting, pastes with less water content than suspended ones, There is a cake-like state in which the water content is less than that in the pasty state, but any state can be used. As the algal bodies used for extraction, it is preferable to use cake-shaped algal bodies. Examples of the dried algal bodies include those obtained by freeze-drying or spray-drying the raw algal bodies obtained by the above method. The processed alga body treated by the mechanical treatment method can be obtained, for example, by subjecting raw alga bodies to ultrasonic treatment or mechanical treatment such as homogenization. The mechanically processed product of algal bodies may be subjected to a drying process thereafter.

  Next, as a method for extracting the ultraviolet absorbing substance from the cultured algal bodies, a method of adding a mixed solvent of water and alcohol to the harvested cultured algal bodies, and stirring and mixing them may be mentioned. The alcohol may be any alcohol that can extract the ultraviolet absorbing material, but methanol or ethanol is preferable, and methanol is most preferable. The mixing ratio of water and alcohol may be any ratio that can extract the ultraviolet absorbing material, but the amount of alcohol is preferably 50 to 100% by weight, and most preferably 75 to 100% by weight. The ratio of the harvested cultured algal bodies to the mixed solvent is preferably 50 to 99% by weight, and particularly preferably 75 to 99% by weight. The extraction temperature may be any temperature as long as the ultraviolet absorbing material can be extracted, but is preferably 0 to 60 ° C, particularly preferably 20 to 40 ° C. The extraction time may be any time as long as the ultraviolet absorbing substance can be extracted, but is preferably 5 minutes to 48 hours, particularly preferably 30 minutes to 2 hours. After the extraction, the suspension of the alga body residue and the mixed solvent containing the ultraviolet absorbing substance is subjected to solid-liquid separation treatment by a method such as centrifugation or filtration to obtain an extract containing the ultraviolet absorbing substance.

  The extract is contaminated with pigments such as chlorophyll having an absorption band in the visible region. In order to use as an ultraviolet absorber, it is preferable to remove these and purify them. The purification of the ultraviolet absorbing substance may be carried out by a known and commonly used treatment method, alone or in combination with column chromatography using activated carbon, gel filtration, ion exchange chromatography, adsorption resin chromatography, high performance liquid chromatography and the like.

Among these, column chromatography using activated carbon, ion exchange chromatography, and adsorption chromatography are preferable, and adsorption chromatography is particularly preferable. Examples of adsorption chromatography include synthetic adsorbents HP20, SP825, SP850, SP200, HP2MG (manufactured by Mitsubishi Chemical Corporation), ODS- _C18 (manufactured by Yamazen Corporation), and SP825 and ODS- _C18 are preferred. ODS-C ₁₈ is particularly preferred. As a specific example using adsorption chromatography, after removing the mixed solvent from the extract using an evaporator or the like, it is dissolved in water and added to a column pre-packed with the adsorption resin, and then the sample is adsorbed to the adsorption resin. After adsorbing on the column, the ultraviolet absorbing material may be eluted with an eluate obtained by mixing water and a solvent such as methanol. The solvent concentration of the eluate may be any as long as the pigments such as chlorophyll do not elute and the ultraviolet absorbing substance elutes, but is preferably 0 to 50% by weight, particularly preferably 5 to 30% by weight. The eluate can be used as an ultraviolet absorbing substance as it is after the solvent is replaced with water or ethanol, or it can be dried and provided as a solid.

  Depending on the application of the UV absorber, the UV absorber obtained at this stage can be used as it is as an UV absorber. However, when used as a UV absorber for cosmetics, foods and drinks, and various industrial products, further fractionation is required. It is preferable to carry out a purification treatment such as high performance liquid chromatography to separate and purify the mycosporin-like amino acid derivative having a glycosyl group of the present invention.

  Separation conditions for preparative high performance liquid chromatography can be generally purified according to the method used for purification of natural products.

  The mycosporin-like amino acid derivative having a glycosyl group can be confirmed in its elution position by measuring the absorption spectrum in the ultraviolet region of the eluate, and the derivative can be fractionated.

  The content derived from a mycosporin-like amino acid having a glycosyl group contained in the thus obtained UV-absorbing substance can be estimated from an elution curve of 320 nm of high-performance liquid chromatography for analysis, etc. Contains 10-300 mg per gram of substance. The ratio of the derivative to the total weight of the UV-absorbing substance varies depending on the UV irradiance and irradiation time during the culture, and the ratio is low in the early culture period when the UV irradiation time is short, and increases in the late culture period when the irradiation time is long. Tend to.

II. Mycosporine-like amino acid derivatives having a glycosyl group Mycosporine-like amino acid derivative having a glycosyl group The mycosporin-like amino acid derivative having a glycosyl group can be obtained by the above production method. Among the isolated ones, specific examples include mycosporin-like amino acid derivatives represented by the following general formula (I).

However, R represents a glycosyl group. Furthermore, examples of the glycosyl group include derivatives that are a glucosyl group, a mannosyl group, and a galactosyl group, and among these, a derivative that is a glucosyl group is preferable. Furthermore, examples of the glucosyl group include derivatives that are a glucopyranosyl group, a mannopyranosyl group, and a galactopyranosyl group, and among these, a derivative that is a glucopyranosyl group is most preferable.

2. Ultraviolet absorbing action The mycosporin-like amino acid derivative having a glycosyl group of the present invention has a strong absorption band in the ultraviolet region having a wavelength of 250 nm to 350 nm.

The ultraviolet absorption ability can be evaluated by the value of the extinction coefficient (E ^1% , 1 cm) (absorbance at a sample concentration of 1.0% and an optical path length of 1 cm). The extinction coefficient can be obtained from an absorption spectrum of an aqueous solution in which a constant weight sample is dissolved. The absorption maximum of the mycosporin-like amino acid derivative having a glycosyl group of the present invention is 320 to 330 nm, and the extinction coefficient (E ^1% , 1 cm) is 500 or more.

3. Moisturizing action In general, when human skin is exposed to sunlight for a long time, collagen fibers in the subcutaneous tissue are broken, pigmentation such as spots and buckwheat, wrinkles due to elastic degeneration, etc. Promoted. In order to prevent such aging, it is important to ensure the moisture retention of the skin in addition to shielding from ultraviolet rays with an ultraviolet absorber.

  The mycosporin-like amino acid derivative having a glycosyl group of the present invention and the ultraviolet absorbing material containing the derivative have a glycosyl group as compared with a mycosporin-like amino acid derivative having no sugar (glycosyl group) which has been reported so far. It has a good moisturizing effect presumed to be caused. On the other hand, among mycosporin-like amino acid derivatives, a mycosporin-like amino acid derivative having a tetrasaccharide has also been reported (T. Rezanka, M. Temina, AG Tolstikov, VM Dembitsky, Folia, Microbiol. 49). (4), 339-352 (2004)). However, this is not only because the amount of saccharides is too much and there are tetrasaccharides, but not only the sticky feeling becomes strong, but also due to the increase in molecular weight, compared with the mycosporin-like amino acid derivative having one glycosyl group of the present invention. The extinction coefficient decreases and the ultraviolet absorption intensity decreases. For this reason, mycosporine-like amino acids having tetrasaccharides are not suitable for cosmetic use compared to the mycosporin-like amino acid derivatives of the present invention having only one sugar.

III. Ultraviolet Absorber As described above, the mycosporin-like amino acid derivative having a glycosyl group of the present invention has a strong ultraviolet absorbing action and an appropriate moisturizing action, so that it is a basic cosmetic, finish cosmetic, and hair as an ultraviolet inhibitor. It can be used by adding to various cosmetics such as cosmetics for soap, soap and shampoo. The obtained cosmetic is useful as an ultraviolet protective cosmetic.

  In addition to the cosmetics field, the mycosporine-like amino acid derivative of the present invention can be used as an ultraviolet absorber by adding it to a use which is usually added in order to exhibit resistance to ultraviolet rays (light resistance). For example, it can be used by adding to photographic materials, heat-sensitive recording materials, light-sensitive heat-sensitive recording materials, inkjet materials, plastics, paints, rubbers, liquid crystal materials, color filters and other industrial products, pharmaceuticals, agricultural chemicals, foods and drinks, etc. .

  In order to describe the contents of the present invention more specifically, the following examples are shown. However, the scope of the present invention is not limited to these examples.

Example 1
1. Algae strain and culture method As the algae strain, the cyanobacteria Nemoptera Nostoc genus Nostoclinkia NIES28 was used. The algae strain was treated with 1 L of MDM medium (KNO ₃ 1 g, MgSO ₄ .7H ₂ O 250 mg, K ₂ HPO ₄ 250 mg, NaCl 100 mg, CaCl ₂ .2H ₂ O 10 mg, Fe solution 1 ml, A5 solution 1 ml, water 998 ml). Inoculate a 1 L glass culture tank with 3 cells. Incubation was carried out by irradiation with four fluorescent lamps (Sanyo FL40SS-VV / 37) at a temperature of 25 ° C. and deep aeration stirring. During this time, the culture solution was maintained at pH 8.0 by appropriately introducing carbon dioxide gas. After 9 days of culture, the algal concentration was 850 mg / L.

2. UV irradiation (1)
Next, four fluorescent lamps (Toshiba FL40S-BLB-A) and five lamps (Sankyo Electric GL40SE) that can irradiate ultraviolet rays in the UVB region are added to the four fluorescent lamps. The other conditions were the same as the above culture conditions. The ultraviolet irradiation intensity was measured using a digital ultraviolet intensity meter (made by Custom Co., Ltd.). The ultraviolet irradiation intensity was 2.5 W / m ² .

3. Measurement of the amount of UV-absorbing substance In addition, since the background of the absorption spectrum is high in a crude extract with many impurities, the difference in absorbance between 330 nm near the absorption maximum and 290 nm, which is the valley of absorbance, is measured, and the optical path length is 1 cm. When the difference in absorbance (330 nm-290 nm) at 0.1 was 0.1, the amount of ultraviolet absorbing substance per 1 ml of solution was defined as 1 unit. 1 ml of the culture solution was centrifuged, 1 ml of methanol was added to the precipitate, and the absorption spectrum of the centrifuged supernatant after stirring and extraction was measured to determine the number of units. FIG. 1 shows the transition of the amount of UV-absorbing substance per hour after cultivation after UV irradiation.
The amount of the UV absorbing substance increased with time until 22 hours of culturing, and thereafter decreased. The amount of the UV absorbing substance in the cultured alga body after 22 hours of culture was about 6000 units / liter of culture solution.

4). Extraction / Purification Method The culture solution 22 hours after the ultraviolet irradiation obtained by the method of Example 1 was centrifuged at 5,000 rpm, the supernatant was removed, and the alga bodies were harvested. 300 ml of methanol (special grade manufactured by Wako Pure Chemical Industries, Ltd.) was added to the alga body (wet weight 9.5 g), and the ultraviolet absorbing material was extracted by stirring for 1 hour. The extracted suspension was centrifuged at 5,000 rpm to obtain a supernatant. The obtained supernatant was purged with methanol using an evaporator and replaced with water. The water-substituted sample was added to a column of a synthetic adsorbent ODS-C ₁₈ (Yamazen Corporation). First, the column was eluted with 3 times the volume of water, and then eluted with 3 times the amount of 25% methanol solution. The eluate was collected and used as an ultraviolet absorbing material. From the absorbance of the ultraviolet absorbing material in a methanol solution, the dry weight of 1000 units of the ultraviolet absorbing material was determined to be about 10 mg.

  Isolation of a mycosporin-like amino acid derivative having a glycosyl group was performed using preparative high-performance liquid chromatography (HPLC apparatus AKTApurifier). HPLC conditions were as follows: Column: Inertsil ODS-3 (20 mm × 250 mm), eluent A: 0.1 M ammonium acetate, eluent B: 74.8 V / V% water, 25 V / V methanol, and 0.2 V / V%. Eluent mixture, gradient elution: (1) eluent A with 2.5 column volume elution, (2) eluent A to eluent B gradient with 3.5 column volume elution, (3) elution Solution B was subjected to 2.0 column volume elution, flow rate: 8 ml / min, detection wavelengths: 330 nm and 270 nm. The sample was concentrated by substituting the above UV-absorbing substance composition with water, filtered, and 2 ml was added.

  The mycosporin-like amino acid derivative having a glycosyl group represented by the general formula (I) was eluted in the vicinity of 300 ml, and showed a single peak in the 320 nm elution curve.

5). Analysis and identification method The mycosporin-like amino acid derivative was identified by HPLC analysis. The chemical structure was determined by LC-MS analysis and NMR analysis.

(1) HPLC analysis Using Shimadzu HPLC analysis system, sample amount: 5 μl, column: Inertsil ODS-3 (6 mm × 259 mm), column temperature: 45 ° C., eluent A: 0.1 M ammonium acetate aqueous solution, eluent B: Mixture of 74.8V / V% water, 25V / V% methanol and 0.2% V / V acetic acid, elution method: After elution with solution A, perform gradient elution from solution A to solution B, Finally, elution was performed only with the B solution. The total elution time is 45 minutes. Flow rate: 1.0 ml / min, detection: PDA method was used, and an elution curve was obtained with an absorbance of 320 nm.

  The mycosporine-like amino acid derivative having a glycosyl group eluted at 9 minutes and showed a single peak. The ultraviolet absorbing substance showed a plurality of peaks, and the amount of the mycosporine-like amino acid derivative having a glycosyl group was determined to be 100 mg / g based on the amount of the ultraviolet absorbing substance from the peak area of the elution time of 9 minutes.

(2) LC-MS analysis The sample was a methanol solution, and the flow injection introduction method was performed under the following conditions. The LC conditions are: HPLC apparatus: HITACHI D-7000 HPLC system, moving bed solvent: methanol, flow rate: 0.1 ml / min, column: Inertosil ODS-2, injection volume: 5-10 μl. The MS conditions are: apparatus: JEOL JMS-LX2000, ionization method: ESI method, polarity: +, −, resolution: 1,500, NEEDLE voltage: 50 V, DESOLVATING temperature: 200 ° C., ORIFICE temperature: 100 ° C.
From this analysis, the molecular weight of the mycosporin-like amino acid derivative contained in the ultraviolet absorbing material was determined to be 450 (FIG. 2).

(3) NMR analysis Add 0.5 ml of DMSO-d _{6 to the} sample, and use NMR apparatus: JEOL JNM-ECA600, 1H, 13C, DEPT-NMR, and HD-COSY, HMQC, HMBC 2D- NMR was measured.

  From each NMR measurement result, it was determined that the chemical structure of the mycosporin-like amino acid derivative contained in the ultraviolet absorbing material has the general formula (I) (FIGS. 3 and 4).

(Examples 2 and 3)
The algae strain and the culture method up to 7 days after the culture were carried out in the same manner as in Example 1. Next, the culture was performed under the irradiation conditions shown below, and the number of UV-absorbing substance units at 22 hours of culture and mycosporin-like having a glucosyl group The amount of amino acid derivative was determined.

1. UV irradiation (2)
Light was irradiated with 4 fluorescent lamps, 3 UVA area lamps (Toshiba FL40S-BLB-A), 3 UVB area lamps (Sankyo Electric GL40SE), and others were cultured in the same manner as in Example 1 Went. The irradiance of ultraviolet rays in the UVB region is 1.5 W / m ² , the number of ultraviolet absorbing substance units after 22 hours of cultivation is about 1200 units / liter of culture solution, the amount of mycosporin-like amino acid derivative having a glycosyl group is ultraviolet absorbing substance The amount was 260 mg / g.

2. UV irradiation (3)
Light was irradiated with 4 fluorescent lamps, 3 UVA area lamps (Toshiba FL40S-BLB-A) and 8 UVB area lamps (Sankyo Electric GL40SE), and the others were cultured in the same manner as in Example 1. Went. The ultraviolet irradiance in the UVB region is 4.0 W / m ² , the number of ultraviolet absorbing substance units after 22 hours of cultivation is about 4000 units / liter of culture solution, and the amount of mycosporin-like amino acid derivative having a glycosyl group is ultraviolet absorbing substance. The amount was 50 mg / g.

  A comparison of the culture results of Examples 1 to 3 is shown in Table 1. Since 1000 units corresponds to about 10 mg of the ultraviolet absorbing substance, the amount of the ultraviolet absorbing substance and the amount of the mycosporin-like amino acid derivative having a glycosyl group in 1 L of the culture solution were calculated. The production amount of the ultraviolet-absorbing substance and the mycosporine-like amino acid derivative having a glycosyl group was the highest under the ultraviolet irradiance of Example 1.

(Measurement Example 1)
The mycosporine-like amino acid derivative having a glycosyl group represented by the general formula (I) obtained in Example 1 was used as a sample for the following measurement.

1. Absorption spectrum and extinction coefficient The absorption spectrum of the sample aqueous solution was measured using Hitachi Spectrum Photometer U-2001. The absorption spectrum is shown in FIG. The absorption spectrum has an absorption maximum in the vicinity of 321 nm and was shown to have the ability to absorb ultraviolet rays in the UVA region effective for sun protection.

The extinction coefficient (E ^1% , 1 cm) was calculated from the absorbance at the maximum value by measuring the absorption spectrum of an aqueous solution of a sample having a constant concentration. The extinction coefficient (E ^1% , 1 cm) at 321 nm of the mycosporin-like amino acid derivative having a glycosyl group was 970.

2. Moisture retention test The moisture retention was determined by measuring the skin moisture content with a moisture meter Corneometer CM825 (Courage + Khazaka electronic). The sample was dissolved in distilled water to prepare a 1% aqueous solution. One drop of this liquid was applied to the inner side of the left upper arm of the person, spread over a 2 cm square part, and inhaled into the skin for 1 minute. After 1 minute, the aqueous solution floating on the surface portion was blotted with paper and measured with a moisture meter after 1 minute.

  The results of measuring the mycosporin-like amino acid derivative (sample 1) having a glycosyl group of the present invention and the mixture of the existing mycosporin-like amino acid derivative porphyra and sinoline (sample 2) are shown in FIG. Sample 1 retained moisture for 40 minutes or more, whereas Sample 2 returned to the amount of moisture before coating in 20 minutes.

  The present invention can be used as an ultraviolet inhibitor in the fields of cosmetics, foods, industrial products and the like.

Relationship between incubation time and amount of UV-absorbing substance The figure showing the LC-MS analysis result of the mycosporin-like amino acid derivative which has a glucosyl group of this invention The figure showing the H-NMR analysis result of the mycosporin-like amino acid derivative which has a glucosyl group of this invention The figure showing the C-NMR analysis result of the mycosporin-like amino acid derivative which has a glucosyl group of this invention Absorption spectrum Moisturizing property test (Sample 1: Mycosporin-like amino acid derivative having a glycosyl group of the present invention, Sample 2: Porphyra and sinoline mixture of existing mycosporin amino acid derivatives)

Claims (10)

The following general formula (I)

A mycosporin-like amino acid derivative having a glycosyl group represented by the formula (wherein R represents a glycosyl group). The mycosporin-like amino acid derivative according to claim 1, wherein R is a glucopyranosyl group. The mycosporin-like amino acid derivative according to claim 1 or 2, wherein the absorption maximum is in the range of 320 to 330 nm, and the extinction coefficient (E ^1% , 1 cm) at the absorption maximum is 500 or more. The ultraviolet absorber containing the said mycosporin-like amino acid derivative of Claim 1. An ultraviolet protective cosmetic comprising the ultraviolet absorber according to claim 4. A method for producing an ultraviolet-absorbing substance comprising a mycosporin-like amino acid derivative having a glycosyl group having a step of culturing an algae belonging to the genus Cyanobacteria Nostococcus while irradiating with active energy rays, wherein irradiation with the active energy rays is performed. Irradiating ultraviolet rays with a wavelength of 280 to 315 nm with an irradiance of 1.5 to 4.0 [W / m ² ]. The mycosporin-like amino acid derivative having a glycosyl group is represented by the following general formula (I)

(However, R represents a glycosyl group) The manufacturing method of the ultraviolet-absorbing substance of Claim 6 represented by these. The method for producing an ultraviolet absorbing material according to claim 7, wherein R is a glucopyranosyl group. The method for producing an ultraviolet absorbing material according to claim 6, wherein the content ratio of the mycosporin-like amino acid derivative having a glycosyl group is in the range of 10 to 300 [mg / g] with respect to the ultraviolet absorbing material. The method for producing an ultraviolet absorbing material according to claim 6, wherein the algae belonging to the genus Cyanobacteria Nostoc is Nostoc linkia.

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